As the proteins (polypeptides) that bind to sugar chains, there have been known sugar-binding proteins, lectins, and lectin-like substances, such as mannose-binding proteins, fibroblast growth factors, and epidermal growth factors. The property of lectin is such that it specifically binds to specific sugar structure. This allows the lectins to be purified using a column on which sugars, sugar chains, or complex carbohydrates are immobilized. Wheat germ lectin and lentil lectin are some of the examples of lectins.
The binding activity of the wheat germ lectin and sugar chain or glycopeptide has been investigated, which suggested that the wheat germ lectin has strong affinity for hybrid type sugar chains among N-glycosidic-binding sugar chains, or sugar chains or glycopeptides having sialic acid (see Non-Patent Publications 1 and 2). It is also known that the wheat germ lectin has stronger binding activity for glycopeptides having a sugar chain structure containing bisecting N-acetylglucosamine (see Non-Patent Publication 3).
It is known that the lentil lectin recognizes the monosaccharides, α-D-mannose and α-D-glucose (see Non-Patent Publication 4). It is also known that the lentil lectin has strong affinity for glycopeptides having a sugar chain in which L-fucose is attached to the α1,6 position of the N-acetylglucosamine residue closest in position to the asparagine of the N-glycosidic-binding sugar chain (see Non-Patent Publications 5 and 6).
Meanwhile, the antibody is known to include a sugar chain structure specific to its Fc region. Purification of antibody is then possible by taking advantage of binding of lectins with the sugar chain. As a method of purifying antibody (particularly, human antibody) using lectins, the method described in Patent Publication 1 is known, for example.
Phylogenetically, chicken is classified lower than mammals; however, they have a sophisticated immune system similar to that of mammals. Specifically, due to the phylogenetic distance from mammals, chicken is useful for the production of specific antibodies against proteins conserved in many mammals. That is, chicken can be used to produce specific antibodies against proteins (antigens), which is difficult to achieve with mice and rats. For example, the antibody against N-acetylneuraminic acid, the antigen as a cancer marker in humans, cannot be produced in animals such as mice or rats because N-glycolylneuraminic acid is present in almost all mammals except humans. Production of the antibody is possible in birds such as chicken, because N-acetylneuraminic acid is not present in birds. Further, production of antibody against the pathogenic prion protein that causes Creutzfeldt-Jakob disease or mad cow disease is also difficult in mammals due to the 90% or greater homology among mammals. The homology between mammal and bird is on the order of 30%, which allows for production of antibody against this particular antigen. In fact, the inventors of the present invention have succeeded in producing chicken monoclonal antibodies against N-acetylneuraminic acid and prion protein by the cell fusion method. Among other advantages of the chicken antibody, use of chicken monoclonal antibody and mammal monoclonal antibody makes it possible to establish a highly sensitive antigen detecting system in which no non-specific reaction occurs, because there is no cross reactivity with the mammal antibody.
As described above, usefulness of chicken antibody (antibody produced by chicken, antibody having the same structure as the antibody produced by chicken), for example, in test and medical applications has been looked into, and establishment of methods for efficiently producing and purifying the chicken antibody is called for. As to the producing method of chicken antibody, progress has been made by techniques such as the cell fusion method and phage display method. However, a further development is needed for the establishment of the purification method.
The chicken antibody does not bind to proteins A and G used as ligands for purifying IgG antibody of mammals. Thus, the method intended for mammals cannot be used directly.
In order to develop a purification technique for chicken antibody, the inventors of the present invention attempted to purify chicken antibody using an affinity column with mouse monoclonal antibody. However, this method failed to purify the chicken antibody. The inventors of the present invention then attempted to purify chicken antibody using gel filtration and ion exchange column. As a result, electrophoretically uniform antibody was successfully purified. However, this method had drawbacks in that it required many steps, was complex, and had a considerably low yield. Among other problems, the purified antibody had a low titer.
In order to overcome such drawbacks, the inventors of the present invention attempted to purify chicken antibody using plant-derived lectins (saxifrage-derived lectin, lentil-derived lectin, and Con A) that specifically bind to the high-mannose-type sugar chains, by taking advantage of the fact that the chicken antibody contains the high-mannose-type sugar chains. However, while the antibody was adsorbed on the saxifrage-derived lectin and the lentil-derived lectin, the antibody could not be eluted. In the case of Con A, the antibody was adsorbed and was eluted with α-methyl glucoside. However, electrophoretically uniform antibody could not be obtained. This was considered to be due to the specific structure of the sugar chain binding to the chicken antibody.
Currently, almost nothing has been revealed as to the sugar chain structure of the chicken antibody (chicken monoclonal antibody). There has been a recent report that the N-asparagine-binding sugar chain (hereinafter, “N-type sugar chain”) of chicken egg yolk antibody (hereinafter, “IgY antibody”) contains glucose on the order of 10% (Ohta, M. et al., Glycoconj. J., 8, 400-413 (1991)). The fact that the N-type sugar chain of mammal IgG antibody does not contain any glucose suggests that the chicken antibody has a unique sugar chain structure. It may therefore be difficult to purify the chicken antibody by directly using the lectins that enable purification of mammal antibodies. It was inferred from the analysis of sugar chain by the inventors of the present invention that the chicken antibody contained both the high-mannose-type sugar chain and the complex type sugar chain, and that more than one glucose was present at the non-reducing end of the sugar chains, or more specifically, one glucose at the non-reducing end of the sugar chain of chicken IgY antibody.
The present invention was made in view of the foregoing problems, and it is an object of the present invention to find a polypeptide (for example, lectin) that binds to a sugar chain, or more specifically a high-mannose-type sugar chain attached to antibody, or more specifically a sugar chain binding to the chicken antibody. The invention also provides a method and means for purifying antibody (particularly, chicken antibody), as representative uses of the polypeptide. It is a further object of the invention to provide a polynucleotide that encodes the polypeptide, an antibody that binds to the polypeptide, and uses of such polynucleotides and antibodies.
[Patent Publication 1]
International Publication WO 02/30954, Pamphlet (published on Apr. 18, 2002)
[Non-Patent Publication 1]
Biochemistry, 16, 4426, 1977
[Non-Patent Publication 2]
The Journal of Biological Chemistry, 254, 4000, 1979
[Non-Patent Publication 3]
Biochemistry, 20, 5894, 1981
[Non-Patent Publication 4]
The Journal of Biological Chemistry, 268, 7668, 1993
[Non-Patent Publication 5]
Carbohydrate Research, 40, 111, 1975
[Non-Patent Publication 6]
Carbohydrate Research, 110, 283, 1975